The Ca2+/calmodulin-regulated phosphatase, calcineurin, is best known for its role in the regulation of cytokine gene expression during T cell activation. However, it is now apparent that this signaling pathway plays a much broader role in the regulation of Ca2+-dependent signaling events in a wide variety of different cell types. As outlined in our preliminary data, we have now evaluated the role of this signaling pathway in the regulation of adipocyte differentiation. We find that the specific calcineurin inhibitors cyclosporin A and FK506 overcome the anti-adipogenic effect of calcium ionophore and PGF2alpha on the differentiation of 3T3-L1 preadipocytes, suggesting that calcineurin is responsible for mediating the previously documented Ca2+-dependent inhibition of adipogenesis. We further find that expression of a constitutively active calcineurin mutant (CNmut) potently inhibits the ability of 3T3-L1 cells to undergo adipocyte differentiation by preventing expression of the critical proadipogenic transcription factors PPARgamma and C/EBPalpha. Finally, we demonstrate that inhibition of endogenous calcineurin activity with either FK506, CsA, or a specific calcineurin inhibitory peptide (VIVIT-GFP) significantly enhances adipocyte differentiation in response to suboptimal adipogenic stimuli. Collectively, these results suggest that calcineurin is an intrinsic negative regulatory component of the normal adipogenic signaling pathway that acts as a Ca2+-dependent molecular rheostat to antagonize adipocyte differentiation. This leads us to propose that the overall degree of activation of the calcineurin signaling pathway in preadipocyte cells is likely to play an important role in determining the efficiency of adipocyte differentiation. Based upon these observations, we hypothesize that calcineurin is likely to play an important role in the regulation of adipogenesis in vivo, and, as such, is likely to influence the accumulation of adipose tissue. In addition to affecting adipocyte differentiation, considerable evidence suggests that calcineurin is also likely to play an important signaling role in mature adipocytes. Accordingly, our primary goals are to determine the mechanisms underlying the inhibitory effect of calcineurin on adipocyte differentiation, to determine the role of calcineurin in mature adipocytes, and to directly test our hypothesis that the calcineurin plays an important role in the regulation of adipose tissue development in vivo. [unreadable] [unreadable]